The overall goal of this research project is to elucidate the molecular mechanisms involved in the transport and toxic actions of heavy metals such as cadmium and lead. This proposal addresses three questions: how do these metals get into cells, where do they go, and how do they act? We previously discovered that Cd2+ enters pituitary cells via voltage sensitive calcium channels and that calcium channel blockers afford protection from cadmium toxicity. Pb2+ has also been shown to enter some cells through voltage-gated calcium channels. We will capitalize on our finding that Cd2+ and Pb2+ increase fluorescence of intracellularly trapped Fura 2 and that Fura 2 and Quin 2 can be used to measure the concentration of intracellular free CD2+ and Pb2+ directly. We will also define the effects of Cd2+ and Pb2+ on calcium homeostasis, distinguishing between effects that are due to metal ion-induced changes in calcium concentration and effects that are due to metal ion substitution in calcium-mediated processes. Initial studies will be performed in endocrine systems where calcium effects have been carefully delineated. Other models, such as neuronal cells with and without active voltage-sensitive calcium channels, will be used later to establish whether the results can be extrapolated to other cell types. Specific aims are as follows. First, the mechanisms of Cd2+ and Pb2+ uptake will be determined. Fluorescence methods will be used to measure free intracellular metal ions. The importance of voltage-sensitive calcium channels in the transport and toxicity of Cd2+ and Pb2+ will be quantified. Other routes of uptake of both free and protein-bound metal ions will be characterized. Second, intracellular free Cd2+ and Pb2+ will be visualized by digital fluorescence imaging techniques. Imaging techniques will be applied to study the localization of metal ions as they enter cells and the localization of free Cd2+ and Pb2+ in chronically exposed cells. The last objective is to determine systematically the effects of Cd2+ and Pb2+ on the function of endocrine cells. The effects of Cd2+ and Pb2+ to disrupt hormone synthesis and secretion stimulated by different signal transduction pathways. The effects of the metal ions on receptors that act via g-proteins (stimulatory and inhibitory adenylate cyclase pathways and phosphoinositide pathways), and on receptors with tyrosine kinase activity will be determined. Finally, the effects of Cd2+ and Pb2+ on hormone synthesis and on nuclear hormone receptors that function as transcription factors will be measured.